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Cryopreservation protocols have been successfully developed for hundreds of species and thousands of genotypes in laboratories around the world. In many of the protocols, the rewarming process occurs in a rewarming solution (RS) with a high concentration of sucrose (0.8-1.2 M). Warming rate and associated conditions influence cell rehydration, loss of accumulated solutes (e.g. cryoprotectants) and recrystallization of small ice crystals in the nuclei. The need for, and effect of, high sucrose concentrations in the RS post-thawing regrowth rate after liquid nitrogen exposure was assessed in the range of 0.0-1.2 M sucrose with a set of 16 potato landraces cryopreserved with the PVS2-droplet vitrification method. The results showed no significant difference for the average recovery rate (81-87%) between sucrose concentrations of 0.3 M to 1.2 M. Fourteen of 16 accessions had their highest recovery rate with sucrose concentrations between 0.3 and 0.9 M. The experimental results were subsequently extended to a genetically diverse set of 85 potato accessions (nine taxa), which demonstrated significantly higher recovery rates of 55-61% with RS sucrose concentrations of 0.3-0.9 M, compared to the sucrose concentrations of 0.0 M (37%) and 1.2 M (44%). Only one of 85 accessions showed its highest recovery rate with the routinely used RS sucrose concentration of 1.2 M. Of all the concentrations tested, 0.6 M sucrose appeared to be the best bet in terms of recovery rates across the genotypes; therefore, our routine protocol has been changed from 1.2 M sucrose to 0.6 M. The specific response to low (0.0 M) and high RS sucrose concentrations (1.2 M) was highly variable within species/subspecies and appears to be genotype specific. Thus, caution should be taken in generalizing experimental cryopreservation results obtained with a limited number of accessions to larger germplasm collections.

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Long-term conservation of Plant Genetic Resources (PGR) is a key priority for guaranteeing food security and sustainability of agricultural systems for current and future generations. The need for the secure conservation of genetic resources collections ex situ is critical, due to rapid and extreme climatic changes which are threatening and reducing biodiversity in their natural environments. The International Potato Center (CIP) conserves one of the most complete and diverse genetic resources collections of potato, with more than 7500 accessions composed of 4900 cultivated potato and 2600 potato wild relative accessions. The clonal conservation of cultivated potato, principally landraces, through in vitro or field collections is indispensable to maintain fixed allelic states, yet it is costly and labor-intensive. Cryopreservation, the conservation of biological samples in liquid nitrogen (-196°C), is considered the most reliable and cost-efficient long-term ex-situ conservation method for clonal crops. Over the last decade, CIP has built one of the largest potato cryobanks worldwide, cyopreserving more than 4000 cultivated potato accessions which represents 84% of the total cultivated potato collection currently conserved at CIP. In approximately, four years the entire potato collection will be cryopreserved. The development of an applied, robust cryopreservation protocol for potato, serves as a model for other clonally maintained crop collections. The CIP cryobank designs experiments with a high number of genetically diverse genotypes (70-100 accessions, seven cultivated species), to obtain reliable results that can be extrapolated over the collection as genotypes can often respond variably to the same applied conditions. Unlike most published reports on cryopreservation of plants, these large-scale experiments on potato are unique as they examine the acclimatization process of in vitro plants prior to, as well as during cryopreservation on up to ten times the number of genotypes conventionally reported in the published literature. As a result, an operational cryopreservation protocol for potato has advanced that works well across diverse potato accessions, not only with reduced processing time and costs, but also with an increased average full-plant recovery rate from 58% to 73% (+LN) for routine cryopreservation. The present article describes the composition of CIP’s cryobank, the cryopreservation protocol, methodology for the dynamic improvement of the operational protocol, as well as data collected on regeneration from long term cryopreserved potatoes.

Root and tuber crops (RTCs) are the second-most important carbohydrate commodity after cereals. Many species of the RTCs are vegetatively propagated, making their shoot tips the preferred material to be conserved for future uses. Shoot tip cryopreservation provides an important tool to support the long-term conservation of plant genetic resources. Over the past four decades, significant efforts have been undertaken to move shoot tip cryopreservation of RTCs from research projects to full-scale implementation in cryobanks. This comprehensive review focuses on the history of cryopreservation protocols developed in RTCs. The encapsulation and vitrification solution-based cryopreservation techniques followed by ultra-rapid freezing and thawing have been highly successful. Additionally, different strategies for improving the cryotolerance of shoot tips have been introduced to further increase post-cryopreservation recovery. Finally, the research conducted to explain the mechanism underlying cryoprotection and differential cryotolerance including the use of histological studies are highlighted.

Cryopreservation is currently the only method which allows long-term conservation of living clonal plant material in the vapor or liquid phase of nitrogen (at −140 to −196 °C) allowing tissue to be viable for decades or perhaps centuries. Specifically, for species with recalcitrant seeds or requiring constant vegetative propagation, it is the method of choice for the long-term conservation of its genetic resources. The protocol described here is a modification of a previously developed plant vitrification solution 2 (PVS2)—droplet vitrification method of potato shoot tips, adapted from Musa species. Utilizing this protocol, the International Potato Center (CIP) has successfully stored in the cryobank more than 3000 cultivated potato accessions, belonging to seven species and nine different taxa [16], originating principally from ten countries in South and Central America. As part of CIP’s quality management system, all vegetative material placed in cryo is routinely subsampled, thawed, and assessed to confirm that whole plantlets can be produced after storage in liquid nitrogen. Complete plant recovery rates of thawed shoot tips range from 20% to 100% (average rate: 60%). This chapter describes the complete set of steps from the routine procedure of cryopreserving potato shoot tips for long-term conservation.

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